When wounded, eukaryotic cells reseal their plasma membrane in a few seconds. This process is essential to avoid loss of cytosolic factors, and for restoring the critical barrier between the intracellular and extracellular environments. Calcium influx through wounds triggers lysosomal exocytosis, an event required for plasma membrane repair. Exocytosis was thought to mediate plasma membrane resealing by addition of an endomembrane patch, or by relieving membrane tension, facilitating spontaneous bilayer restoration. However, it recently became clear that calcium influx also triggers the repair of lesions caused by pore-forming proteins. When inserted in the plasma membrane, pore-forming proteins generate stable lesions that cannot be resealed by a patch, or by reducing membrane tension. An investigation of this process revealed that calcium influx in injured cells markedly stimulates endocytosis, with a kinetics that coincides with cell resealing. Additional results suggested that trans-membrane pores and mechanical lesions are removed from the plasma membrane by endocytosis, and that this process requires exocytosis of a lysosomal enzyme, acid sphingomyelinase. These new findings represent a major conceptual advance in our understanding of plasma membrane repair, since they indicate that the role of lysosomal exocytosis is to release a critical hydrolase that acts on the cell surface, and not to add a patch or relieve membrane tension. We hypothesize that calcium entry triggers exocytosis of lysosomal acid sphingomyelinase, which cleaves sphingomyelin at the cell surface, generating ceramide and inducing formation of endosomes that carry the lesions into the cells for degradation. To test this hypothesis, we will pursue two specific aims: 1) Determine if exocytosis of lysosomal acid sphingomyelinase during plasma membrane wounding leads to ceramide generation and endosome formation, and whether this injury repair pathway is present in muscle fibers;2) Determine the intracellular fate of the endosomes generated during plasma membrane wounding with pore-forming toxins. In addition to clarifying how cells survive attack by membrane damaging agents produced by pathogens, this project will provide new insight on mechanisms underlying the pathology of serious human diseases, including lysosomal storage diseases and muscular dystrophy.

Public Health Relevance

Plasma membrane repair is essential for cellular survival after injury. The goal of this proposal is to investigate the mechanism by which mammalian cells remove wounds on their membrane by endocytosis. The results will clarify how cells survive attack by toxins produced by pathogens, and will increase our knowledge of defects leading to serious human diseases including lysosomal storage diseases and muscular dystrophy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064625-11
Application #
8494635
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2002-08-01
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
11
Fiscal Year
2013
Total Cost
$361,878
Indirect Cost
$120,626
Name
University of Maryland College Park
Department
Anatomy/Cell Biology
Type
Schools of Earth Sciences/Natur
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742
Castro-Gomes, Thiago; Corrotte, Matthias; Tam, Christina et al. (2016) Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes. PLoS One 11:e0152583
Fernandes, Maria Cecilia; Corrotte, Matthias; Miguel, Danilo C et al. (2015) The exocyst is required for trypanosome invasion and the repair of mechanical plasma membrane wounds. J Cell Sci 128:27-32
Miller, Heather; Castro-Gomes, Thiago; Corrotte, Matthias et al. (2015) Lipid raft-dependent plasma membrane repair interferes with the activation of B lymphocytes. J Cell Biol 211:1193-205
Corrotte, M; Castro-Gomes, T; Koushik, A B et al. (2015) Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses. Methods Cell Biol 126:139-58
Andrews, N W; Corrotte, M; Castro-Gomes, T (2015) Above the fray: Surface remodeling by secreted lysosomal enzymes leads to endocytosis-mediated plasma membrane repair. Semin Cell Dev Biol 45:10-7
Andrews, N W; Perez, F (2015) The plasma membrane repair shop: Fixing the damage. Semin Cell Dev Biol 45:1
Andrews, Norma W; Almeida, Patricia E; Corrotte, Matthias (2014) Damage control: cellular mechanisms of plasma membrane repair. Trends Cell Biol 24:734-42
Hissa, Barbara; Pontes, Bruno; Roma, Paula Magda S et al. (2013) Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes. PLoS One 8:e82988
Flannery, Andrew R; Renberg, Rebecca L; Andrews, Norma W (2013) Pathways of iron acquisition and utilization in Leishmania. Curr Opin Microbiol 16:716-21
Corrotte, Matthias; Almeida, Patricia E; Tam, Christina et al. (2013) Caveolae internalization repairs wounded cells and muscle fibers. Elife 2:e00926

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